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Appl. Environ. Microbiol. doi:10.1128/AEM.00418-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Polyphasic approach to bacterial dynamics during the ripening of Spanish farmhouse cheese by culture-dependent and culture-independent methods

Dpto. de Microbiología, Facultad de Ciencias, Fuentenueva s/n, 18071 Granada, Spain

^* To whom correspondence should be addressed. Email: mmartine{at}ugr.es.

	Abstract

We have studied the dynamics of the microbial population during the ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during the ripening period of the Spanish goats' milk cheese using Lactococcus lactis and Streptococcus thermophilus as starter cultures were analysed. All bacterial isolates were clustered by randomly amplified polymorphic DNA (RAPD) and identified by 16S ribosomal DNA gene sequencing, species-specific PCR and multiplex PCR.

Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were non-starter lactic-acid bacteria (NSLAB), Lactobacillus paracasei being the most abundant. Other Lactobacillus species such as Lb. plantarum and Lb. parabuchneri were also detected at the beginning and end of ripening respectively. Non-lactic-acid bacteria (NLAB), mainly Kocuria and Staphylococcus strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal-temperature-gradient gel-electrophoresis (TTGE) provided us with a more precise estimation of the distribution of bacteria and enabled us to detect Lb. curvatus and the starter bacteria Streptococcus thermophilus and Lactococcus lactis, which were not isolated. Surprisingly, the bacterium most frequently found by culture-dependent analysis, Lb. paracasei, was scarcely detected by this molecular approach. Finally, we studied the composition of the Lactobacilli and their evolution by length-heterogeneity PCR.